The long-term objective of this proposed research is to determine the biological functions and to elucidate the molecular mechanisms of DNA topoisomerases, which play a key role in various biological processes of DNA metabolism. These processes include or may include DNA replication, transcription, recombination, chromosome condensation and decondensation, nucleosome assembly, virus encapsidation and DNA transposition. In this proposed research, I will focus my studies on one functional aspect of DNA topoisomerases, namely the initiation of DNA replication. A general model is proposed for the mechanism of initiation of replication forks. Negative supercoiling of DNA is proposed to be essential for activating the origins. The role of those DNA topoisomerases that are involved in the initiation of DNA replication is presumably to provide negative supercoiling. In the cases of certain large genomes, such as T4 bacteriophage DNA and eukaryotic DNA, only limited domains (spanning the origins) are proposed to be supercoiled by origin-specific gyrases. This model will be tested both in T4 bacteriophage system and in Drosophila early embryos. In T4 system, the fork initiation process will be reconstructed in vitro by using seven purified T4 coded proteins, which are essential for at least the elongation mode of DNA replication, and T4 DNA topoisomerase is likely to be the origin-specific gyrase of T4 bacteriophage. Enzymes with similar properties to T4 DNA topoisomerase will be searched for in Drosophila early embryos. An in vitro Drosophila DNA replication system will be developed and the fork initiation mechanism of eukaryotic DNA replication will be similarly approached.